Process for measuring low cadmium levels in blood and other biological specimens

ABSTRACT

A process for measuring low levels of cadmium in blood and other biological specimens is provided without interference from high levels of alkali metal contaminants by forming an aqueous solution and without contamination by environmental cadmium absent the proteins from the specimen, selectively removing cadmium from the aqueous solution on an anion exchange resin, thereby removing the alkali metal contaminants, resolubilizing cadmium from the resin to form a second solution and analyzing the second solution for cadmium, the process being carried out in a cadmium-free environment.

CONTRACTUAL ORIGIN OF THE INVENTION

The U.S. Government has rights in this invention pursuant to ContractNo. W-31-109-ENG-38 between the U.S. Department of Energy and theUniversity of Chicago representing Argonne National Laboratory.

BACKGROUND OF THE INVENTION

This invention relates to the measurement of cadmium in biologicalspecimens and more particularly to the detection and measurement ofultratrace cadmium levels in blood and other biological fluids.

Measurement of cadmium in blood, urine, tissue and other biologicalspecimens at low levels is important for a number of reasons. Cadmiumhas a long biological half-life and the toxic effects of exposure arecumulative. Chronic exposure to cadmium can irreversibly damage thekidneys. Cadmium is now considered to be a potential carcinogen. Also,recent biomedical research suggests that low-level cadmium exposure fromsmoking may contribute to osteoporosis.

Cadmium in addition to lead may be found in the working environment, inambient air, in drinking water, in tobacco smoke and in food. Certainfoods may be the major contributors to human exposure to cadmium.Smoking may be an additional significant source of cadmium exposure.Because cadmium accumulates in humans from different sources, biologicalmonitoring has become recognized as a desired way to estimate the totalexposure and risks associated with cadmium.

The measurement of the level of toxic metals in human body fluids isoften the best way of evaluating undue exposure, whether the source beindustrial, environmental or iatrogenic. To be of any use in diagnosingand monitoring exposed individuals, analytical results generated must bereliable (i.e., accurate), and reproducible with time.

The determination of cadmium in biological samples is of considerableinterest to health and research organizations. Studies have beenconducted to measure and monitor cadmium (and other toxic substances) inbody fluids. One study, as reported in "An Interlaboratory ComparisonProgramme For Several Toxic Substances in Blood and Urine" byJean-Philippe Weber, in The Science of the Total Environment, (1988) 71,111-123, was directed to the measurement of cadmium (and other toxicsubstances) in blood and urine as performed by a number of laboratories.Consistency in the measurements of cadmium even at levels above 5 μg/Lwas difficult as indicated by the reported coefficients of variation inthe order of 30-40%, with higher values for lower concentrations.

The most widely used analytical technique for measuring cadmium in bloodand urine samples involves the steps of deproteination and directanalysis by electrothermal atomic absorption spectrophotometry (ETAAS).In general, this technique has provided acceptable consistency ofresults with samples having a cadmium concentration above about 5 μgCd/L.

One reason for the difficulty in measuring cadmium at levels of ≦5 μg/Lis that the deproteination or wet-ashing step in the above describedtechnique removes cadmium from its organic environment but does notseparate it from inorganic constituents. Sodium and potassiumconcentrations in normal blood are generally 10⁶ times greater thancadmium. When using electrothermal atomic absorption spectrophotometry(ETAAS) with deuterium background correction for the analysis of cadmiumin blood, this inorganic environment contributes significantly to theinaccuracy and imprecision of the measurement. Another difficultyassociated with measuring cadmium in blood arises from the presence ofcadmium as an ubiquitous environmental contaminant. Control or avoidanceof cadmium contamination by glassware, plastic ware, reagents and air isof utmost importance for proper accuracy.

In order to evaluate cadmium exposures in an environmental setting, itis important to measure cadmium at low levels (i.e., below about 0.5μg/L) in blood, urine and other biological specimens. Seawater is alsoan important medium for measuring cadmium content since cadmium may havean adverse effect on developing fish embryos. Data on cadmium levels,particularly in body fluids, and changes in those levels related toexposure to special environmental conditions, could be of considerableimportance in providing early indications of medical problems. Since thetoxic effects of exposure are reported to be cumulative, information ofthis type could also be particularly important in association with testson young persons to determine early adverse exposure.

Accordingly, one object of this invention is a method of testingbiological specimens for low cadmium concentrations. Another object ofthe invention is a method of testing biological fluids containing highvalues of alkali metal contaminants for cadmium. Yet another object ofthe invention is a method for testing biological fluids for low cadmiumconcentration with improved accuracy. These and other advantages of theinvention will become apparent from the following description.

SUMMARY

Briefly, the invention is directed to the measurements of cadmium inbiological fluids containing levels of alkali metal contaminants whichwould interfere with accurate measurements for cadmium and moreparticularly to measurements in body fluids such as blood, plasma andurine at cadmium levels below about 5 μg/L. The measurement is carriedout by the steps of forming an aqueous solution having an acid pH andessentially free of insolubles from the biological fluid with HCl toconvert the cadmium to an anioninc cloride selectively removing cadmiumfrom the aqueous solution on an anion exchange resin, resolubilizingcadmium from the resin to form a second solution and analyzing forcadmium in the second solution advantageously by electrothermal atomicabsorption spectrophotometry. It is important that the environmentincluding the reagents, column with anion exchange resin and processequipment such as glass and plasticware be essentially cadmium-free(i.e., contributing a total of less than about 0.1 ng Cd to the assay)and that the processing of the samples be carried out under a hood toreduce air contaminants.

For particularly low cadmium concentrations (i.e., below about 0.5μg/L), the invention advantageously includes the step of increasing thecadmium content of the second solution by depositing cadmium on theresin from a larger volume of the first solution. By this technique, acadmium concentration of 0.2 μg/L associated with the first aqueoussolution may be increased to about 0.8 μg/L by contacting the resin with8 mL of the first solution and resolubilizing the cadmium into 2 mL ofthe second solution for analysis.

Since it is important to use reagents and equipment essentially Cd-free,the invention also includes a kit comprising the reagents, equipment,one or more packages containing the reagents and equipment in anessentially cadmium-free environment with instructions descriptive ofthe method of the invention.

The separation of cadmium on the anion exchange resin will also normallyinclude zinc which has a chromatographic behavior similar to cadmium.Since the signal from the ETAAS is reduced by the presence of zinc, theinvention includes a corrective factor in the standards and the sampleto offset the effect of zinc.

The invention has a number of advantages. Measurements of cadmiumconcentrations in the order of 0.2 μg/L may be carried out withacceptable accuracy and lack of contamination by Cd in the atmosphere.These measurements are also associated with improved reliability basedon the coefficient of variation (CV). Use of the invention typicallywill remove about 97 wt. % of the contaminants from a sample of bloodwhile providing a solution for cadmium analysis containing greater than94 wt. % of the original cadmium.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is an elution profile of ¹⁰⁹ Cd separated from whole blood ( ,plasma (∇), urine ( ), and pure acids (o).

FIG. 2 is a graph showing the effect of zinc on the peak area absorbanceof cadmium using ETAAS analysis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention is particularly useful to carry out measurements ofcadmium in biological samples and particularly those associated withbody fluids such as blood, urine and the like. For simplification, thefollowing description will be directed to measurement in samples ofblood, plasma and urine. However, it should be recognized that analysisof other biological samples of cadmium in such fluids as seawater may beconducted with the described technique without undue experimentation.

The process for measuring cadmium in the samples primarily involves thesteps of forming an aqueous salt solution with HCl to convert thecadmium to an anionic chloride, the solution including any inorganiccontaminants from the sample, selectively removing cadmium from thesolution on an anion exchange resin, resolubilizing cadmium on the resinto form a second solution and analyzing the second solution to provide avalue representative of the cadmium content in the sample.

In the process, cadmium is separated from the primary contaminants priorto the analysis. These contaminants are principally inorganic andespecially the alkali metal contaminants at significant levels in thebody fluid. Advantageously, the invention will provide a solution foranalysis in which greater than 99 wt. % of the alkali metal contaminantshave been removed and a background absorption unit with respect to theanalysis below 1 unit. Table I below provides a representative bloodcomposition before separation and the cadmium sample after separation.As shown, zinc remains with the cadmium.

                  TABLE I                                                         ______________________________________                                        Concentrations of Cadmium and Major Elements in the                           Supernatant of Deproteinated Whole Blood and in the Cadmium                   Fraction After Anion-Exchange Separation                                               Deproteinated                                                                           Cadmium fraction                                                    Blood (μg/ml)                                                                        (μg/ml)                                                 ______________________________________                                        Ca         13.4        0.01                                                   Cu         0.16        <0.01                                                  Fe         3.3         0.1                                                    K          625         0.5                                                    Mg         11.0        <0.01                                                  Na         505         0.5                                                    Zn         1.43        1.47                                                   Cd         0.00014     0.00013                                                ______________________________________                                    

Since the contaminants are removed prior to the analysis, testing ofblanks and standards by ETAAS provide extremely low reference levels.Under these circumstances, detection limits in the order of 0.01-0.2 μgCd/L may be achieved depending on the particular fluid and sample size.The detection limit is defined as 3×SD (standard deviation) of the blankvalue.

The process is also capable of measuring cadmium at low levels in thebiological samples with reasonable precision. The coefficient ofvariation (CV) is used as a measure of precision in the tests and isdefined as the standard deviation/mean as a percentage. Based on 2 mlsamples, measurements of cadmium at levels from 1 μg Cd/L and above andparticularly 1-5 μg Cd/L provide CV values of 15-20% and below for about3 replicate analyses. For cadmium contents in the general range of0.3-1.0 μg Cd/L, CV values are generally in the order of 20% and belowfor about 3 analyses. For low values of 0.05-0.3 μg Cd/L, CV values willgenerally be greater than 20% although generally below about 25%. Atthese low values, larger sample volumes (i.e., 4 ml or 8 ml) may be usedwith the CV values being reduced to about 20-25%. In general, CV valuesbelow about 25% are achieved by use of the invention.

In the process, it is important that the samples be prepared in aCd-free environment which limits the external cadmium effect in theassay to a total of about 0.1 ng Cd, particularly when cadmiumconcentrations are below about 0.1 μg Cd/L. This requires the use ofextremely pure water and acids having cadmium concentrations well below1 ppb. Acid-washed glassware and metal-free plasticware are also used.Sample preparation is under a Class 100 hood. Gloves are used inhandling the requipment and the test tubes are covered during transfer.A plastic cover is provided over the ETAAS unit. In addition, it isusually desirable to run a blank and test standard every three samplesto check the ETAAS for calibration and accuracy.

The use of a ¹⁰⁹ Cd "spike" may be useful in the analysis technique toincrease the sensitivity of the assay. FIG. 1 shows the generaldistribution of ¹⁰⁹ Cd from five different portions (1 ml) of a sampleprepared with ¹⁰⁹ Cd on the anion exchange resin. As shown, about 55 wt.% of ¹⁰⁹ Cd is in the second portion (or second ml) of the sample. Intesting for cadmium in a sample, ¹⁰⁹ Cd may be added to the sample, anda selected portion eluted from the anion exchange resin may be analyzed,with the measured value of the stable cadmium (other than ¹⁰⁹ Cd)increased by a factor equal to ¹⁰⁹ Cd added/¹⁰⁹ Cd in the analyzedportion. The result will be an increase in assay sensitivity.

In contrast to the other contaminants, zinc is also deposited on theresin and requires a corrective factor to obtain an accurate value forcadmium. FIG. 2 shows the effect of zinc on the measurements, with avalue of about 1.4-1.5 mg Zn/L and above requiring an increase in theorder of 14-18%. Advantageously, the presence and content of zinc in asample is separately determined by flame atomic absorption or the likeprior to the cadmium analysis with the appropriate Zn being added to theblank and standard fluids used in calibrating the ETAAS unit and forcomparison purposes.

Since this process is capable of concentrating low concentrations of Cdin biological fluids, the routine measurement of plasma Cdconcentrations has become possible. Recent Cd exposure (within 12-24hours) appears as an increase in plasma Cd concentrations. By 24 hoursafter a single subcutaneous exposure, nearly all of the Cd in blood isbound within the blood cells. The ratio of Cd in plasma to Cd in wholeblood therefore provides a measure of a person's very recent exposure.The plasma Cd concentration is important because Cd in plasma, ratherthan that bound to blood cells, is generally believed to be responsiblefor the delivery of Cd to peripheral organs such as kidney, liver, andbone.

The results of plasma and blood Cd measurements in beagles exposed to 15ppm Cd in water using this process are shown in Table II below. Theratios of plasma/blood Cd are shown in the last column and range from2.6-16.5%. One likely benefit of determining the plasma/blood Cd ratiowill be its use by scientists studying Cd to provide new insights fordose-response relationships and the mechanism of Cd toxicity to variousorgans.

                  TABLE II                                                        ______________________________________                                        Cadmium Concentration (μg/liter) in Plasma (Cd-P) Versus                   Blood (Cd-B) of Canis familiaris Exposed to Cadmium in Water                  Sample   Cd-P.sub.E                                                                            Cd-P.sub.H Cd-B Cd-P.sub.H /Cd-B                             ______________________________________                                        A        0.72    0.46       8.98 0.051                                        B        2.35    2.46       21.98                                                                              0.112                                        C        0.96    1.37       8.87 0.154                                        D        1.87    1.98       11.98                                                                              0.165                                        E        1.58    1.13       13.71                                                                              0.082                                        F        0.48    0.57       12.12                                                                              0.047                                        G        0.48    0.52       19.58                                                                              0.026                                        ______________________________________                                    

Advantageously, the analysis for cadmium is by an atomic absorptiontechnique and preferably by electrothermal atomic absorptionspectrophotometry (ETAAS). The principle of this technique is based uponthe absorption of light at element-specific wavelengths (resonance line)for neutral atoms in the ground state. Solutions containing cadmium areplaced onto a piece of pyrolytically coated graphite and dried. Thegraphite is heated very quickly by passing an electrical current acrossit. When the temperature of the graphite reaches the boiling point ofthe element or a salt of it, the element forms a gaseous cloud in thelight path. The amount of light that is absorbed is proportional to thequantity of ground state neutral atoms formed from the element depositedonto the graphite. Absorption of light by other elements or molecules inthe gas cloud produced during atomization (background absorbance) canintroduce significant errors in the analysis. For this reason, a secondbeam of light is passed through the atomic vapor to measure lightabsorption due to the background material. This procedure is calleddeuterium background correction and works well when backgroundabsorption is kept below 1.0 absorbance unit. A more sophisticated formof background correction is available which has been reported to becapable of correcting background absorbances greater than 2.0 absorbanceunits. It is called Zeeman background correction and uses strongmagnetic fields to intermittently polarize the light. In practice,atomic absorption spectrophotometers are calibrated with known standardsso that the absorbance readings obtained from unknown samples can bedirectly converted to concentration units.

Particularly with body fluids of low cadmium content, the processadvantageously includes a step of increasing the cadmium content in thesolution (second solution) of resolubilized cadmium. A larger sample ofthe body fluid may be used to provide a larger volume (i.e., 8 ml) ofthe aqueous solution used to treat the anion exchange resin. The cadmiumwould then be resolubilized in a 2 ml solution with the cadmium contentbeing increased by a factor of four permitting more accurate analysis.

In carrying out the test procedures associated with the invention, it isparticularly important that the reagents, the glass and plastic ware andthe samples are free from contamination by cadmium in the air, water, orother sources. The assay requires extremely pure water and concentratedacids having cadmium concentrations well below 1 ppb. Equipment used forthe analysis of cadmium in blood includes a standard atomic absorptionspectrophotometer equipped with electrothermal atomization and deuteriumbackground correction, centrifuge, laminar flow hood or equivalentquality air, properly acid-washed glassware and plasticware, and liquidmeasurement devices capable of accurate and precise measurements in therange of 5 μl to 10 ml and anion exchange columns. Advantageously,Cd-free equipment is provided as a kit with a package or packages toavoid contamination and instructions. Representative instructions are asfollows:

KIT INSTRUCTIONS Special Precautions

Three reagents in this kit contain corrosive acid (hydrochloric acid,nitric acid, and sulfuric acid) and should be handled with the properprotective equipment (i.e., rubber gloves, laboratory coat, safetygoggles). During all manipulations, the samples should be protected fromairborne cadmium contamination by working in a Class 100 air safety hoodor by covering the samples with screw caps or cadmium-free plastic wrap.Because cadmium is a ubiquitous environmental contaminant, painstakingcare must be taken to avoid cadmium's introduction from such sources asair, handling, and glassware. Cigarette smoke contains cadmium, and thisanalysis should not be attempted unless the laboratory environment issmoke-free. Samples should be handled using rubber gloves that have beenrinsed with deionized water. Any plasticware (i.e, pipette tips,beakers, etc.) should be demonstrated free from cadmium contamination orshould be rinsed with dilute nitric acid followed by a deionized waterrinse. Glassware used should be leached in approximately 1N HNO₃overnight, rinsed with cadmium-free water, dried and covered withplastic wrap to prevent airborne contamination. Special attention shouldbe paid to the way the sample is collected. Vacutainers used for blooddraw and storage should be tested routinely for cadmium contamination.Although the stainless-steel needles used for blood collection are anunlikely source of contamination, they should be tested if all othersources of contamination have been ruled out.

Kit Contents:

Reagents:

(1) 170 ml conc HNO₃

(2) 250 ml conc HCl

(3) 10 ml 10% H₂ SO₄

(4) 1 gal. (3.7 L) ultra-pure H₂ O

(5) 2 ml of Zn standard (1000 μg/ml,Cd-free)

Components:

(1) 55 g Anion Exchange resin

(2) 100 glass frits

(3) 50 polypropylene chrom columns

(4) 160 15 ml metal-free centrifuge tubes with screw cap

Equipment needed (but not supplied):

1. Centrifuge which will hold 15 ml centrifuge tubes and providecentrifugal force of approximately 1300×g.

2. Pipetting devices which use disposable plastic tips and are capableof delivering volumes in the range of 20 μl-10 μl with good precision.

3. Source of Class 100 air (HEPA filtered air, such as obtained from abiological safety cabinet).

4. Cd-free plastic pipette tips (polypropylene tips without any dyeadded to the formulation will normally suffice; some manufacturers offermetal-free plastic products, such as Elkay Products, Inc., Shrewsbury,Mass.).

5. Atomic Absorption Spectrophotometer equipped with electrothermalatomization.

The process for measuring cadmium in blood or other biological materialrequires a sample of a body fluid such as blood, plasma or urine.Extraction of cadmium is carried out by diluting the whole blood withhigh resistance water and removing proteins and fat by the addition ofnitric acid followed by centrifugation. Nitric acid is preferred overhydrochloric since hydrochloric might attack the protein content of thesample. The resultant supernatant liquid contains cadmium in theremaining inorganic salts. This supernatant is further acidified with 2NHCl to convert the cadmium to an anionic chloride. In the next step,cadmium is separated from the remaining salts using anion exchangechromatography. Cadmium behaves as an anion in 2N HCl and is boundtightly by a strong anion exchange resin. The remaining salts inbiological specimens such as sodium, potassium, calcium, magnesium, donot form anions and pass through the anion exchange column. Cadmium andzinc (and possibly mercury) bind to the columns and are eluted in asmall volume of 1N HNO₃ with 100% recovery of the cadmium. These may bereferred to as metals in column 2b of the Periodic Table with atomicweights of 30-80. Column elution is thus a simple two-step batchprocess. The 2N HCl step concentrates the cadmium on the top of theanion exchange resin, allowing as much sample to be applied as needed toachieve the desired sensitivity. For example, increasing the sample from2 ml to 8 ml can increase the measurement limit by a value in the orderof 10 times. In this manner, detection limits in the order of 0.2 ppb Cdmay result in lower values of 0.02 ppb Cd. Following the column elution,the cadmium released from the column in a nearly salt-free solution isconcentrated in a small volume in preparation for analysis. Sulfuricacid is added to the column eluate to a level of 0.1% (vol/vol) in orderto reduce the volatilization of cadmium in the analysis step. Cadmium isthen analyzed by standard atomic absorption analysis usingelectrothermal atomization with deuterium background correction.

Accordingly, the invention may be considered as a process for analyzingcadmium in biological specimens by a combination of steps which includeextracting cadmium and other water soluble salts in a water solution,separating cadmium from essentially all of the other salts by the use ofan anion exchange resin and detecting cadmium in the remaining solutionby atomic absorption analysis. Advantageously, the invention utilizes azinc correction to compensate for the reduced signal caused by thepresence of zinc. Also, advantageously, the invention includes a kitwhich includes acid reagents with levels of cadmium below 1 ppb, highresistance water with cadmium content below 1 ppb, glass and plasticvessels having cadmium content below 1 ppb, and within appropriatepackaging containing instructions.

The algorithm used to convert the concentration of thecadmium-containing fractions after anion exchange back to cadmiumconcentration for a sample is described below. Typically, the volumefrom the column is 5 ml and the initial volume of the sample is 2 ml.The algorithm is applicable to the analysis of samples of blood, plasmaand urine.

    ______________________________________                                        Cd (analyzed) ng/ml × volume* = ng Cd recovered (R)                                                  [1]                                              ng Cd.sub.(R) × 6/5 (dilution by HCl) × 8/5 (reciprocal           fraction of aqueous solution-SN1) =                                                                        [2]                                              ng Cd corrected                                                               ng Cd corrected divided by (initial volume of                                                              [3]                                              sample used) = ng Cd/ml = ppb                                                 ______________________________________                                         *volume collected from anion exchange column                             

The following examples are provided for illustrative purposes and arenot intended to be restrictive as to the scope of the invention:

EXAMPLES I-III

Samples of blood from a human donor were analyzed for cadmium content.In the test, each sample of whole blood was diluted 1:4 (2 mL wholeblood+6 mL 1N HNO₃) in Cd-free test tubes (either acid washed glass ormetal free polypropylene) and vortexed for 30 sec. After standing forabout 15 min., the samples were centrifuged for about 10 min. at 1300×g.The samples were covered to prevent airborne contamination. Aftercentrifugation, the surface fats and lipoproteins were removed byadsorption to plastic pipette tips prior to pipetting the firstsupernatant (SN1). In preparation for column chromatography, 5 mL of adeproteinated SN1 was transferred to a clean test tube and 1.0 mL of12.8M hydrochloric acid was added. The samples were vortexed again.After a second 10-min. centrifugation at 1300×g (to remove any suspendedparticulates), a 5 mL sample (SN2) was added directly to theequilibrated anion exchange column. This resin was identified by thesupplier, BIO-RAD Laboratories of Richmond, Calif., as a strongly basicanion exchanger with quaternary ammonium functional groups attached tothe styrene divinylbenzene copolymer lattice. It is further identifiedas an AG1 resin which is resistant to oxidation. The column was rinsedwith 3 mL 2N HCl. Cadmium was eluted or resolubilized from the columnwith 1N HNO₃. The first 2 mL went to waste (based on a pretestdetermination that the first 2 mL advanced the cadmium to the bottom ofthe column), and the following 5 mL were collected in an acid-cleanedborosilicate test tube for cadmium analysis. Five microliters ofconcentrated sulfuric acid was added to prevent loss of Cd as Cd(NO₃)during drying or ashing. Original sample volumes at 2 ml, 4 ml and 8 mlwere subjected to the above assay steps and were analyzed for cadmium byelectrothermal atomic absorption spectrophotometry (ETAAS).

The analysis by ETAAS was carried out by the conventional technique asdescribed previously. The technique is also described in Stoeppler andBrandt (1980), Fresenius Z. Anal. Chem. 300, 372-380. Compensation forbackground was provided by deuterium background correction. Theinstrument was calibrated with known standards for cadmium. Since thepresence of zinc affected the peak area absorbance of cadmium analyzedby ETAAS, zinc was added to the standards used to generate the standardcurve for cadmium analysis. The cadmium concentration of unknown sampleswas compared to standards prepared in 1N HNO₃, 0.1% H₂ SO₄ v/v, andcontaining 1-6 mg Zn/L.

The test results for blood are shown in Table III below. As shown, three2 mL samples were analyzed for cadmium with the results being 0.57±0.004μg/L and a CV of 10%. For the two 4 mL samples, the results were0.62±0.05 μg/L and a CV of 8%. For the 8 mL samples, two samplesprovided consistent values of 0.45±0.02 μg/L with a CV of 6%. One 8 mLsample tested at 0.72 μg/L with the significant difference beingattributed to operation of the furnace associated with the ETAAS test(spurious contamination). The CV for the three samples was 30%.

                  TABLE III                                                       ______________________________________                                        Effect of Blood Sample Volume on Cadmium Recovery                                          Cadmium                                                          Sample       concentration                                                    volume       (μg/liter)                                                                             CV (%)                                               ______________________________________                                        2 ml         0.57 ± 0.04(3)                                                                         10                                                   4 ml         0.62 ± 0.05(2)                                                                          8                                                   8 ml         0.54 ± 0.16(3)                                                                         30                                                   ______________________________________                                    

The results show that in general the concentration obtained isessentially independent of sample volume, particularly at concentrationsin the order of 0.10 μg Cd/L and above.

EXAMPLES IV-VI

Plasma samples were analyzed using the same procedures as described forwhole blood except as modified by deproteinating the plasma by directaddition of concentrated HNO₃ [0.3 mL concentrated HNO₃ +6 mL plasma (5%HNO₃)] with 4 mL of the deproteinated supernatant (SN1) being mixed with0.8 mL of 12.8 M HCl before adding 4 mL of the sample to the columncontaining the anion exchange resin. The second centrifugation step wasalso not required for the plasma samples.

Tests for cadmium were carried out with 2 mL, 4 mL and 8 mL of plasmawith two tests at each volume. The results are shown in Table IV below.The results (mean±standard error) at 2 mL level were 0.10±0.05 μg/L witha CV of 70%, at 4 mL were 0.10±0.02 μg/L with a CV of 25%, and at 8 mLwere 0.06±0.01 with a CV of 20%. As shown, the standard error and CVdecreased from 70% with the small volume to 20% with the larger volumesof test sample when the cadmium concentration in the test sample was low(0.06 μg Cd/L).

                  TABLE IV                                                        ______________________________________                                        Effect of Plasma Sample Volume on Assay Precision                                          Cadmium                                                          Sample       concentration                                                    volume       (μg/liter)                                                                             CV (%)                                               ______________________________________                                        2 ml         0.10 ± 0.05(2)                                                                         70                                                   4 ml         0.10 ± 0.02(2)                                                                         25                                                   8 ml         0.06 ± 0.01(2)                                                                         20                                                   ______________________________________                                    

The donor was the sam for the blood and plasma samples of Exapmles I-VIas shown in Table III-IV. Therefore, information on the ratio of plasmaCd to blood Cd is significant and shows values ranging from 11-16%.

EXAMPLES VII-VIII

Urine samples (5 ml) were analyzed for cadmium using the proceduresdescribed above except as modified by direct acidification of urine withconcentrated NHO³ (instead of dilution with 1N HNO₃) with the resultsfor five tests provided in Table V below.

                                      TABLE V                                     __________________________________________________________________________    Assay Characteristics - Precision and Accuracy                                        Cadmium                       Detection                                       concentration                                                                              Cadmium                                                                             Volume                                                                             Recovery                                                                            limit                                           (μg/liter)                                                                         CV (%)                                                                             added (ng)                                                                          (ml) %     (μg Cd/liter)                        __________________________________________________________________________    Intra-assay                                                                   Blood   0.57 ± 0.10(7)                                                                     17   --    2-8  --    0.21 (2 ml)                                                                   0.10 (4 ml)                                                                   0.05 (8 ml)                                     0.32 ± 0.03(4)                                                                     9    --    8    --    0.01 (8 ml)                             Plasma  0.09 ± 0.01(5)                                                                     11   --    8    --    0.02 (8 ml)                             Plasma + Spike                                                                        0.27 ± 0.02(5)                                                                     8    1.6   8    90    0.02 (8 ml)                             Urine   0.19 ± 0.05(5)                                                                     26   --    5    --    0.02 (5 ml)                             Urine + Spike                                                                         0.76 ± 0.03(5)                                                                     4    2.5   5    114   0.02 (5 ml)                             Interassay                                                                    Blood   0.61 ± 0.08(7)                                                                     13   --    2    --    0.21 (2 ml)                             Blood + Spike                                                                         1.06 ± 0.10(6)                                                                     9    1.0   2    90                                            Blood + 5.32 ± 0.27(6)                                                                     5    10.0  2    94                                            __________________________________________________________________________

As shown in Table V the urine samples contained 0.19±0.05 μg Cd/L with aCV of 26%. The samples spiked with Cd (0.5 μg Cd/L) tested at 0.76±0.03μg Cd/L with a CV of 4%. Comparisons between the two tests show an assayvalue for the spike of 0.57 μg Cd/L (0.76-0.19) which is withinacceptable limits.

The foregoing description of embodiments of the invention has beenpresented for purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdisclosed. Other modifications and variations are possible in light ofthe above teaching.

The embodiments of this invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A process for measuringhalf microgram levels of cadmium in biological fluids containing atleast one alkali metal contaminant, comprisingthe steps of forming anaqueous solution of cadmium and the contaminant from a sample of thefluid converting the cadmium to anionic cadmium chloride selectivelyremoving cadmium apart from a substantial portion of the contaminantfrom the aqueous solution on an anion exchange resin, resolubilizingcadmium from the resin to form a second solution, and analyzing forcadmium in the second solution, the process being carried out in anessentially cadmium-free environment.
 2. The process of claim 1 whereinthe analysis for cadmium is carried out by electrothermal atomicabsorption.
 3. The process of claim 1 including the step of increasingthe cadmium content of the second solution by increasing the volume ofthe aqueous solution compared to the volume of the second solution. 4.The process of claim 1 wherein the fluid is blood, plasma or urine. 5.The process of claim 1 wherein the sample contains zinc and the processincludes the step of increasing the analyzed value by a predeterminedamount to correct for the limiting effect of Zn on the analyzed valuefor cadmium.
 6. The process of claim 1 including the steps of adding apredetermined amount of ¹⁰⁹ Cd to the sample before resolubilizing thesample cadmium from the resin and analyzing for ¹⁰⁹ Cd as well as samplecadmium to provide a correlation between the analyzed sample cadmiumwith sample cadmium in the second solution.
 7. The process of claim 1including the step of selecting respective volumes of the aqueous andsecond solutions and the number of samples to provide a measure ofcadmium content in the fluid with a coefficient of variation less thanabout 25%.
 8. The process of claim 1 including the step of analyzing forblood and plasma to form a ratio separately for cadmium a patient usefulin detecting recent exposure to harmful levels of cadmium.
 9. A processfor measuring half microgram levels of cadmium in biological fluidscontaining at least one alkali metal contaminant, comprisingthe steps offorming an aqueous solution of cadmium and the contaminant from a sampleof the fluid, selectively removing cadmium apart from a substantialportion of the contaminant from the aqueous solution on an anionexchange resin, resolubilizing cadmium from the resin to form a secondsolution, and analyzing for cadmium in the second solution, the processbeing carried out in an essentially cadmium-free environment, thecadmium content of the second solution being increased by increasing thevolume of the aqueous solution compared to the volume of the secondsolution, and a corrective factor for Zn being applied to correct forthe limiting value of Zn on the analyzed value for cadmium.